ABSTRACT Enhancers are non-coding DNA elements near genes that positively regulate gene expression. Because enhancers often act in a cell type specific manner, they confer biological specificity on gene regulation. Common genetic variation within enhancers appears to be a prevalent means of trait association. Super-enhancers are a recently described set of enhancers highly enriched for regulatory elements of cell lineage-defining genes and genetic variants related to lineage-specific disease susceptibility. However the structural determinants of super-enhancer function remain poorly understood. Enhancers are typically defined by their correlated biochemical features or ectopic potential in reporter assays. Nonetheless the gold-standard test of enhancer activity is loss-of-function mutation to discern the requirement for regulatory sequences in their natural chromosomal environment. Our group has developed a Cas9-mediated in situ saturating mutagenesis technique that allows for high-resolution, high-throughput functional analysis of enhancers in the native genomic setting. The assay involves design and synthesis of guide RNAs saturating a region of interest followed by pooled lentiviral CRISPR screening. This method when coupled with genomic target deep sequencing allows for an evaluation nearing nucleotide resolution of the functional sequences critical for enhancer function. In this proposal, we apply this technique to systematically perturb twenty erythroid-specific super-enhancers of key erythroid genes. We extend the Cas9 mutagenesis strategy by making use of a variant Cas9 to allow for increased genome editing resolution. In addition, we take a variant-informed guide design approach to improve mutagenesis even in the face of natural genomic variation. We utilize bioinformatic methods to identify essential sequences, predict interacting transcription factors, and refine models to estimate enhancer activity. We use biochemical techniques to determine the occupancy of transcription factors and chromatin regulators at required sequences. We focus on defining and testing super-enhancer looping interactions in which the key transcription factors GATA1 and TAL1 participate. Finally we employ prospective reverse genetics to validate both cis-acting sequences and trans-acting factors necessary for super-enhancer function. These studies will help determine emergent cooperative properties of clustered components within super-enhancers. We expect these experiments to inform a further understanding of crucial aspects of erythropoiesis as well as fundamental mechanisms of gene regulation.